This proposal investigates cell envelope stress responses in Bacillus subtilis, a model organism for the Gram positive bacteria. Antibiotics and other agents that impair the synthesis or function of the cell membrane or peptidoglycan cell wall are of critical importance in clinical medicine. Exposure to sub-lethal levels of antibiotics activates the transcription of sets of genes (cell envelope stress responses) that serve to remodel the cell wall and membrane. In B. subtilis, these stress responses are coordinated by alternative sigma subunits for RNA polymerase of the extracytoplasmic function (ECF) subfamily. This proposal is focused on defining the role of ECF sigma factors in controlling adaptive changes in cell envelope function. One focus will be on the roles of induced lipid biosynthesis pathways and proteins in the genesis and function of lipid rafts. A second focus will be to discover the roles of a newly defined second messenger, cyclic-di-AMP, that is required for cell envelope homeostasis. Using a combination of classical and molecular genetics, genomics, transcriptomics, and biochemical approaches, these studies will provide insights into cell envelope synthesis and function, and reveal novel mechanisms of antibiotic resistance.